Pneumococcal polysaccharides and their use in immunogenic polysaccharide-carrier protein conjugates

ABSTRACT

The present invention provides capsular polysaccharides from  Streptococcus pneumoniae  serotypes identified using NMR. The present invention further provides polysaccharide-protein conjugates in which capsular polysaccharides from one or more of these serotypes are conjugated to a carrier protein such as CRM197. Polysaccharide-protein conjugates from one or more of these serotypes may be included in multivalent pneumococcal conjugate vaccines having polysaccharides from multiple additional  Streptococcus pneumoniae  serotypes.

FIELD OF INVENTION

The present invention provides purified capsular polysaccharides fromStreptococcus pneumoniae serotypes 23A and 23B, andpolysaccharide-protein conjugates having polysaccharides from one ormore of these serotypes. Polysaccharide-protein conjugates from one ofmore of these serotypes may be included in multivalent pneumococcalconjugate vaccines.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae, one example of an encapsulated bacterium, is asignificant cause of serious disease world-wide. In 1997, the Centersfor Disease Control and Prevention (CDC) estimated there were 3,000cases of pneumococcal meningitis, 50,000 cases of pneumococcalbacteremia, 7,000,000 cases of pneumococcal otitis media and 500,000cases of pneumococcal pneumonia annually in the United States. SeeCenters for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep1997, 46(RR-8):1-13. Furthermore, the complications of these diseasescan be significant with some studies reporting up to 8% mortality and25% neurologic sequelae with pneumococcal meningitis. See Arditi et al.,1998, Pediatrics 102:1087-97.

The multivalent pneumococcal polysaccharide vaccines that have beenlicensed for many years have proved invaluable in preventingpneumococcal disease in adults, particularly, the elderly and those athigh-risk. However, infants and young children respond poorly tounconjugated pneumococcal polysaccharides. Bacterial polysaccharides areT-cell-independent immunogens, eliciting weak or no response in infants.Chemical conjugation of a bacterial polysaccharide immunogen to acarrier protein converts the immune response to a T-cell-dependent onein infants. Diphtheria toxoid (DTx, a chemically detoxified version ofDT) and CRM197 have been described as carrier proteins for bacterialpolysaccharide immunogens due to the presence of T-cell-stimulatingepitopes in their amino acid sequences.

The pneumococcal conjugate vaccine, Prevnar®, containing the 7 mostfrequently isolated serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) causinginvasive pneumococcal disease in young children and infants at the time,was first licensed in the United States in February 2000. Followinguniversal use of Prevnar® in the United States, there has been asignificant reduction in invasive pneumococcal disease in children dueto the serotypes present in Prevnar®. See Centers for Disease Controland Prevention, MMWR Morb Mortal Wkly Rep 2005, 54(36):893-7. However,there are limitations in serotype coverage with Prevnar® in certainregions of the world and some evidence of certain emerging serotypes inthe United States (for example, 19A and others). See O'Brien et al.,2004, Am J Epidemiol 159:634-44; Whitney et al., 2003, N Engl J Med348:1737-46; Kyaw et al., 2006, N Engl J Med 354:1455-63; Hicks et al.,2007, J Infect Dis 196:1346-54; Traore et al., 2009, Clin Infect Dis48:S181-S189.

Prevnar 13® is a 13-valent pneumococcal polysaccharide-protein conjugatevaccine including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A,19F and 23F. See, e.g., U.S. Patent Application Publication No. US2006/0228380 A1, Prymula et al., 2006, Lancet 367:740-48 and Kieningeret al., Safety and Immunologic Non-inferiority of 13-valent PneumococcalConjugate Vaccine Compared to 7-valent Pneumococcal Conjugate VaccineGiven as a 4-Dose Series in Healthy Infants and Toddlers, presented atthe 48^(th) Annual ICAAC/ISDA 46^(th) Annual Meeting, Washington D.C.,Oct. 25-28, 2008. See, also, Dagan et al., 1998, Infect Immun. 66:2093-2098 and Fattom, 1999, Vaccine 17:126.

S. pneumoniae has been categorized into more than ninety serotypes basedon the structure of the capsular polysaccharide. A list of knownpneumococcal capsular polysaccharide structures is provided in Geno,2015, Clinical Microbiology Reviews 28:871-899. Serotype 23A wasdescribed in Italian Patent No. IT 1418572 B1 but no structure wasprovided.

The current multivalent pneumococcal conjugate vaccines have beeneffective in reducing the incidence of pneumococcal disease associatedwith those serotypes present in the vaccines, e.g., 23F. However, theprevalence of the pneumococci expressing serotypes not present in thevaccine has been increasing. Moreover, pneumococcal conjugate vaccineswhich include 23F do not afford cross protection to serotypes 23A and23B. Accordingly, there is a need to identify and characterize emergingpneumococcal serotypes for inclusion in future vaccines.

SUMMARY OF THE INVENTION

The present invention provides purified capsular polysaccharides fromStreptococcus pneumoniae serotypes 23A and 23B, and polysaccharideprotein conjugates having these serotypes. The present invention isbased, in part, on the structural identification of capsularpolysaccharides from these serotypes.

Accordingly, in one embodiment, the present invention provides apolysaccharide with one of the following repeating units:

A polysaccharide from Streptococcus pneumoniae serotype 23A can berepresented by

where n represents the number of repeating units.

A polysaccharide from Streptococcus pneumoniae serotype 23B can berepresented by

where n represents the number of repeating units.

In certain embodiments, the polysaccharide has between 10 and 5,000repeating units. In certain aspects, the polysaccharide has between 50and 3,000, 100 and 2,500, or 100 to 2,000 repeating units.

In certain embodiments, the polysaccharide has a molecular weight from50 kDa to 4,000 kDa. In certain aspects, the polysaccharide has amolecular weight from 80 kDa to 2,000 kDa, or 100 kDa to 1,500 kDa.

The present invention further provides activated polysaccharidesproduced from any of the above embodiments wherein the polysaccharide isactivated with a chemical reagent to produce reactive groups forconjugation to a linker or carrier protein. In certain embodiments, theactivation of S. pneumoniae serotype polysaccharide 23A occurs on theα-Rhap or β-Glcp. In certain embodiments, the activation of S.pneumoniae serotype polysaccharide 23B occurs on the β-Glcp or β-Rhap.In one aspect of this embodiment, activation of S. pneumoniae serotypepolysaccharide 23B occurs at greater than 90%, 95%, or 99% on theβ-Rhap. In certain embodiments, the polysaccharide is activated withperiodate. In certain aspects of this embodiment, the activation of theserotype 23A polysaccharide occurs on the 2^(nd) or 3^(rd) carbonposition of α-Rhap or β-Glcp or the activation of the serotype 23Bpolysaccharide occurs on the 2^(nd) or 3^(rd) carbon position of β-Glcpor β-Rhap. In one sub-aspect of this aspect, perodiate activation of theserotype 23B polysaccharide occurs at greater than 90%, 95%, or 99% onthe β-Rhap.

The present invention further provides polysaccharide-protein conjugatesin which polysaccharides or activated polysaccharides as provided forabove are conjugated to a carrier protein. In certain aspects, thecarrier protein is selected from CRM197, diphtheria toxin fragment B(DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment Cof TT, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, andexotoxin A from Pseudomonas aeruginosa. In one specific aspect, thecarrier protein is CRM197.

In certain aspects, the polysaccharide-protein conjugates are preparedusing reductive amination chemistry under aqueous conditions or in anaprotic solvent such as dimethyl sulfoxide (DMSO). In a specific aspect,the polysaccharide-protein conjugates are prepared using reductiveamination chemistry in DMSO.

In one embodiment, the present invention provides a multivalentimmunogenic composition comprising unconjugated polysaccharides orpolysaccharide-protein conjugates from one or more of Streptococcuspneumoniae serotypes 23A and 23B, and unconjugated polysaccharides orpolysaccharide-protein conjugates from one or more of Streptococcuspneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N,9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18B, 18C, 19A, 19F, 20,21, 22A, 22F, 23F, 24B, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F, and38. In one subembodiment, a multivalent immunogenic compositioncomprises unconjugated polysaccharides or polysaccharide-carrier proteinconjugates but not both. In one subembodiment, a multivalent immunogeniccomposition comprises a mixture of unconjugated polysaccharides orpolysaccharide-carrier protein conjugates. In certain subembodiments, amultivalent immunogenic composition of the invention has up to 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, or 90 serotypes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B depicts graphical representations of the repeating unitstructure of S. pneumoniae serotype 23A (A) and 23B (B) polysaccharides.The activation sites available for periodate are shown with arrows. Foran activated polysaccharide, not all of the repeating unit's activationsites are activated. This reflects the possible activation of theserotype 23B polysaccharide on the 2nd or 3rd carbon position of β-Rhap.

FIG. 2A-B depict the 600 MHz one-dimensional 1H NMR spectrum of thecapsular polysaccharide from S. pneumoniae serotype 23A (A) and 23B (B)in deuterium oxide (D₂O) at 50° C. Signals arising from internalstandards (DMSO and DSS-d₆), residual water (HOD) and other residualcomponents from the purification process; ethanol (EtOH), isopropanol(IPA) and acetate, are marked. Minor signals marked by * are due to S.pneumoniae cell wall residuals such as C-polysaccharide and/orpeptidoglycans.

FIGS. 3A-B depict the one-dimensional (1D) ¹H NMR identity region to beused for serotype identifications of S. pneumoniae serotype 23A (A) and23B (B). Signal positions of each anomeric proton of the repeating unitfrom each monosaccharide residue is marked.

FIGS. 4A-B depict partial two-dimensional (2D) ¹H-¹³C multiple bondcorrelation NMR spectrum of S. pneumoniae serotype 23A (A) and 23B (B)establishing covalent linkages between sugar residues in the repeatingstructure. Correlation establishing glycosidic linkages are labeled inthe figure.

FIGS. 5A-B depicts establishment of phosphodiester linkages in thecapsular polysaccharide repeating unit of S. pneumoniae serotype 23A (A)and 23B (B).

FIG. 6: 600 MHz one-dimensional ¹H NMR spectrum of oxidized and TSCderivatized capsular polysaccharide from S. pneumoniae serotype 23B indeuterium oxide (D₂O) at 25° C. The insert is an expansion of the iminesignals formed by derivatization with thiosemicarbazide.

FIG. 7: 2D TOCSY of spectrum of oxidized and TSC derivatized capsularpolysaccharide from S. pneumoniae serotype 23B. The correlation signalsbetween 7.36-7.40 ppm peaks with rhamnose CH₃ peaks (˜1.32 ppm) arecircled. Insert is the structure for capsular polysaccharide from S.pneumoniae serotype 23B, the periodate activation site has beenindicated with an arrow.

FIG. 8A-B: 2D gCOSY (A) and NOESY (B) of spectrum of oxidized and TSCderivatized capsular polysaccharide from S. pneumoniae serotype 23B.

FIG. 9: ELISA IgG antibody titers (post-dose 2) for rabbits immunizedwith S. pneumoniae monovalent polysaccharide serotypes conjugated toCRM197 and formulated with aluminum phosphate adjuvant (APA). Error barsrepresent the geometric mean+95% confidence interval.

FIG. 10: Serotype specific OPA titers (post-dose 2) for rabbitsimmunized with S. pneumoniae monovalent polysaccharide serotypesconjugated to CRM197 and formulated with aluminum phosphate adjuvant(APA). Error bars represent the geometric mean+95% confidence interval.

FIGS. 11A-B: A. ELISA PnPs23A IgG antibody titers; and B. S. pneumoniaepolysaccharide serotype 23A OPA titers (pre-immune, post-dose 1 (PD1)and post-dose 2 (PD2)) for rabbits immunized with S. pneumoniaemonovalent polysaccharide serotypes 23A, 23B or 23F conjugated to CRM197and formulated with aluminum phosphate adjuvant (APA). Error barsrepresent the geometric mean+95% confidence interval.

FIGS. 12A-B: A. ELISA PnPs23B IgG antibody titers; and B. S. pneumoniaepolysaccharide serotype 23B OPA titers (pre-immune, post-dose 1 (PD1)and post-dose 2 (PD2)) for rabbits immunized with S. pneumoniaemonovalent polysaccharide serotypes 23A, 23B or 23F conjugated to CRM197and formulated with aluminum phosphate adjuvant (APA). Error barsrepresent the geometric mean+95% confidence interval.

FIGS. 13A-B: A. ELISA PnPs 23F IgG antibody titers; and B. S. pneumoniaepolysaccharide serotype 23F OPA titers (pre-immune, post-dose 1 (PD1)and post-dose 2 (PD2)) for rabbits immunized with S. pneumoniaemonovalent polysaccharide serotypes 23A, 23B or 23F conjugated to CRM197and formulated with aluminum phosphate adjuvant (APA). Error barsrepresent the geometric mean+95% confidence interval.

FIG. 14 shows serotype specific (S. pneumoniae serotypes 16F, 23A, 23B,24F, 31) pre-immune, PD1 and PD2 geometric mean antibody titers forrabbits immunized with a multivalent pneumococcal conjugate vaccine (2μg/PnPs). Error bars represent 2 standard errors of the geometric meantiter of each serotype (X-axis).

FIG. 15 shows serotype specific (S. pneumoniae serotypes 16F, 23A, 23B,24F, 31) pre-immune, PD1 and PD2 OPA dilution titers for rabbitsimmunized with a multivalent pneumococcal conjugate vaccine (2 μg/PnPs).Symbols indicate the individual titers and error bars represent the 95%confidence intervals (CIs) of the geometric mean titers (GMTs). *p<0.05,**p<0.01, ***p<0.001, ns=not significant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the identification of novelpneumococcal polysaccharide structures by NMR technology. It is believedthat the structures provided herein are the first identification or thefirst correct identification of these S. pneumoniae serotypes 23A and23B.

The S. pneumoniae serotypes 23A and 23B polysaccharides were producedfrom their respective strains and purified. The produced (and purified)polysaccharides were used to generate individual Ps-CRM197 conjugates.S. pneumoniae serotypes 23A and 23B have a unique polysaccharidestructure, which results in a conjugate production process. Theresulting conjugates were demonstrated to be immunogenic in animalstudies.

As used herein, the term “polysaccharide” (Ps) is meant to include anyantigenic saccharide element (or antigenic unit) commonly used in theimmunologic and bacterial vaccine arts, including, but not limited to, a“saccharide”, an “oligosaccharide”, a “polysaccharide”, a“liposaccharide”, a “lipo-oligosaccharide (LOS)”, a “lipopolysaccharide(LPS)”, a “glycosylate”, a “glycoconjugate”, a “derivatized or activatedpolysaccharide or oligosaccharide”, and the like. Unless otherwisespecified, the polysaccharide nomenclature used herein follows theIUB-IUPAC Joint Commission on Biochemical Nomenclature (JCBM)Recommendations 1980. See JCBN, 1982, J. Biol. Chem. 257:3352-3354.

As used herein, “immunogenic composition” refers to a compositioncontaining an antigen, such as a bacterial capsular polysaccharide or apolysaccharide-protein conjugate, that has the ability to elicit animmune response in a host such as a mammal, either humorally orcellularly mediated, or both. The immunogenic composition may serve tosensitize the host by the presentation of the antigen in associationwith MHC molecules at a cell surface. In addition, antigen-specificT-cells or antibodies can be generated to allow for the futureprotection of an immunized host. Immunogenic compositions thus canprotect the host from infection by the bacteria, reduced severity, ormay protect the host from death due to the bacterial infection.Immunogenic compositions may also be used to generate polyclonal ormonoclonal antibodies, which may be used to confer passive immunity to asubject. Immunogenic compositions may also be used to generateantibodies that are functional as measured by the killing of bacteria ineither an animal efficacy model or via an opsonophagocytic killingassay.

As used herein, the term “isolated” in connection with a polysacchariderefers to isolation of S. pneumoniae serotype specific capsularpolysaccharide from purified polysaccharide using purificationtechniques known in the art, including the use of centrifugation, depthfiltration, precipitation, ultrafiltration, treatment with activatecarbon, diafiltration and/or column chromatography. Generally anisolated polysaccharide refers to partial removal of proteins, nucleicacids and non-specific endogenous polysaccharide (C-polysaccharide). Theisolated polysaccharide contains less than 10%, 8%, 6%, 4%, or 2%protein impurities and/or nucleic acids. The isolated polysaccharidecontains less than 20% of C-polysaccharide with respect to type specificpolysaccharides.

As used herein, the term “purified” in connection with a bacterialcapsular polysaccharide refers to the purification of the polysaccharidefrom cell lysate through means such as centrifugation, precipitation,and ultra-filtration. Generally, a purified polysaccharide refers toremoval of cell debris and DNA.

As used herein, the term “Mw” refers to the weight averaged molecularweight and is typically expressed in Da or kDa. Mw takes into accountthat a bigger molecule contains more of the total mass of a polymersample than the smaller molecules do. Mw can be determined by techniquessuch as static light scattering, small angle neutron scattering, X-rayscattering, and sedimentation velocity.

As used herein, the term “Mn” refers to a number average molecularweight and is typically expressed in Da or kDa. Mn is calculated bytaking the total weight of a sample divided by the number of moleculesin the sample and can be determined by techniques such as gel permeationchromatography, viscometry via the (Mark-Houwink equation), colligativemethods such as vapor pressure osmometry, end-group determination orproton NMR. Mw/Mn reflects polydispersity.

As used herein, the term “molar ratio” is a fraction typically expressedas a decimal to the tenths or hundredths place. For example, a molarratio of from 0 or 0.1 to 1.0 expressed in tenths will include any of0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.

As used herein, the abbreviation “PnPs” refers to pneumococcalpolysaccharide.

As used herein, the term “comprises” when used with the immunogeniccomposition of the invention refers to the inclusion of any othercomponents (subject to limitations of “consisting of” language for theantigen mixture), such as adjuvants and excipients. The term “consistingof” when used with the multivalent polysaccharide-protein conjugatemixture of the invention refers to a mixture having those particular S.pneumoniae polysaccharide protein conjugates and no other S. pneumoniaepolysaccharide protein conjugates from a different serotype.

As used herein, the phrase “activation site” on a sugar means that thesite can be chemically modified to form a reactive group. Activationsite takes into account the preferred tendency of an activation agent toreact at a specific site.

As used herein, the phrase “activated polysaccharide” refers to apolysaccharide that has been chemically modified to form reactive groupsin a polysaccharide chain. An activated polysaccharide does notnecessarily mean that all the available activation sites have beenchemically modified.

As used herein, the phrase “extent of activation” on a polysaccharidechain refers to the overall ratio between the number of activatedchemical group to the number of repeat units on the polysaccharidechain.

Unless otherwise specified, all ranges provided herein are inclusive ofthe recited lower and upper limits.

Two additional members of S. pneumoniae serogroup 23, for which nostructure or composition information has been available, have beenidentified. The serotype 23B polysaccharide has the same backbone as theserotype 23F polysaccharide, but is missing the pendant α-Rhap. Incomparison to the serotype 23F polysaccharide, the serotype 23Apolysaccharide possesses a shorter backbone and a longer side chain.

The identification of the structure for these serotypes may allow theirincorporation into pneumococcal vaccines, either unconjugated or as apolysaccharide-protein conjugate. Conjugate vaccines comprisingstreptococcal and pneumococcal Ps are well-known in the art. See e.g.,U.S. Pat. Nos. 6,248,570; 5,866,135; and 5,773,007.

Capsular Polysaccharides

Capsular polysaccharides from Streptococcus pneumoniae from theserotype(s) of the invention can be prepared by standard techniquesknown to those skilled in the art. For example, polysaccharides can beisolated from bacteria and may be sized to some degree by known methods(see, e.g., European Patent Nos. EP497524 and EP497525); and preferablyby microfluidisation accomplished using a homogenizer or by chemicalhydrolysis. In one embodiment, S. pneumoniae strains corresponding toeach polysaccharide serotype are grown in a soy-based medium. Theindividual polysaccharides are then purified through standard stepsincluding centrifugation, precipitation, and ultra-filtration. See,e.g., U.S. Patent Application Publication No. 2008/0286838 and U.S. Pat.No. 5,847,112. Polysaccharides can be sized in order to reduce viscosityand/or to improve filterability of subsequent conjugated products.Chemical hydrolysis may be conducted using acetic acid. Mechanicalsizing may be conducted using High Pressure Homogenization Shearing.

In some embodiments, the purified polysaccharides before conjugationhave a molecular weight of between 5 kDa and 4,000 kDa. Molecular weightcan be calculated by size exclusion chromatography (SEC) combined withmultiangle light scattering detector (MALS) and refractive indexdetector (RI). In other such embodiments, the polysaccharide has anaverage molecular weight of between 10 kDa and 4,000 kDa; between 50 kDaand 4,000 kDa; between 50 kDa and 3,000 kDa; between 50 kDa and 2,000kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,000 kDa; between50 kDa and 750 kDa; between 50 kDa and 500 kDa; between 80 kDa and 2000kDa; between 100 kDa and 4,000 kDa; between 100 kDa and 3,000 kDa; 100kDa and 2,000 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa;between 100 and 400 kDa; between 200 kDa and 4,000 kDa; between 200 kDaand 3,000 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,500kDa; between 200 kDa and 1,000 kDa; or between 200 kDa and 500 kDa. Incertain embodiments, the polysaccharide from serotype 23A has an averagemolecular weight of between 75 kDa and 200 kDa. In certain embodiments,the polysaccharide from serotype 23B has an average molecular weight ofbetween 150 kDa and 250 kDa.

In certain embodiments, the S. pneumoniae serotypes 23A or 23Bpolysaccharide has between 10 and 5,000 repeating units. In certainaspects, the polysaccharide has between 50 and 3,000, 100 to 2,500, or100 to 2,000. In certain embodiments, the polysaccharide from serotype23A has between 97 and 260 repeating units. In certain embodiments, thepolysaccharide from S. pneumoniae serotype 23B has between 195 and 324repeating units.

Carrier Protein

Polysaccharides from one or more of the serotypes can be conjugated to acarrier protein (“Pr”) to improve immunogenicity in children, theelderly and/or immunocompromised subjects. Where more than one serotypeis used in a multivalent composition, the serotypes may be prepared withthe same carrier protein or different carrier proteins. Each capsularpolysaccharide of the same serotype is typically conjugated to the samecarrier protein.

In a particular embodiment of the present invention, CRM197 is used as acarrier protein. CRM197 is a non-toxic variant of diphtheria toxin (DT).The CRM197 carrier protein is a mutant form of DT that is renderednon-toxic by a single amino acid substitution in Fragment A at residue52. In one embodiment, the CRM197 carrier protein is isolated fromcultures of Corynebacterium diphtheria strain C7 (β197) grown incasamino acids and yeast extract-based medium. In another embodiment,CRM197 is prepared recombinantly in accordance with the methodsdescribed in U.S. Pat. No. 5,614,382. Typically, CRM197 is purifiedthrough a combination of ultra-filtration, ammonium sulfateprecipitation, and ion-exchange chromatography. In some embodiments,CRM197 is prepared in Pseudomonas fluorescens using Pfenex ExpressionTechnology™ (Pfenex Inc., San Diego, Calif.).

Other suitable carrier proteins include additional inactivated bacterialtoxins such as DT, Diphtheria toxoid fragment B (DTFB), TT (tetanustoxid) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g., asdescribed in International Patent Application Publication No. WO2004/083251), E. coli LT (heat-labile enterotoxin), E. coli ST(heat-stable enterotoxin), and exotoxin A from Pseudomonas aeruginosa.Bacterial outer membrane proteins such as outer membrane complex c(OMPC), porins, transferrin binding proteins, pneumococcal surfaceprotein A (PspA; See International Application Patent Publication No. WO02/091998), pneumococcal adhesin protein (PsaA), C5a peptidase fromGroup A or Group B streptococcus, or Haemophilus influenzae protein D,pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63; 2706-13)including ply detoxified in some fashion for example dPLY-GMBS (SeeInternational Patent Application Publication No. WO 04/081515) ordPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE and fusions of Phtproteins for example PhtDE fusions, PhtBE fusions (See InternationalPatent Application Publication Nos. WO 01/98334 and WO 03/54007), canalso be used. Other proteins, such as ovalbumin, keyhole limpethemocyanin (KLH), bovine serum albumin (BSA) or purified proteinderivative of tuberculin (PPD), PorB (from N. meningitidis), PD(Haemophilus influenzae protein D; see, e.g., European Patent No. EP 0594 610 B), or immunologically functional equivalents thereof, syntheticpeptides (See European Patent Nos. EP0378881 and EP0427347), heat shockproteins (See International Patent Application Publication Nos. WO93/17712 and WO 94/03208), pertussis proteins (See International PatentApplication Publication No. WO 98/58668 and European Patent No.EP0471177), cytokines, lymphokines, growth factors or hormones (SeeInternational Patent Application Publication No. WO 91/01146),artificial proteins comprising multiple human CD4+ T cell epitopes fromvarious pathogen derived antigens (See Falugi et al., 2001, Eur JImmunol 31:3816-3824) such as N19 protein (See Baraldoi et al., 2004,Infect Immun 72:4884-7), iron uptake proteins (See International PatentApplication Publication No. WO 01/72337), toxin A or B of C. difficile(See International Patent Publication No. WO 00/61761), and flagellin(See Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used ascarrier proteins.

Other DT mutants can also be used as the carrier protein, such asCRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218:3838-3844);CRM9, CRM45, CRM102, CRM103 and CRM107 and other mutations described byNicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, MaecelDekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Serand/or Ala 158 to Gly and other mutations disclosed in U.S. Pat. No.4,709,017 or 4,950,740; mutation of at least one or more residues Lys516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed inU.S. Pat. No. 5,917,017 or 6,455,673; or fragment disclosed in U.S. Pat.No. 5,843,711.

Where multivalent vaccines are used, a second carrier protein can beused for one or more of the antigens. The second carrier protein ispreferably a protein that is non-toxic and non-reactogenic andobtainable in sufficient amount and purity. The second carrier proteinis also conjugated or joined with an antigen, e.g., a S. pneumoniaepolysaccharide to enhance immunogenicity of the antigen. Carrierproteins should be amenable to standard conjugation procedures. In oneembodiment, each capsular polysaccharide not conjugated to the firstcarrier protein is conjugated to the same second carrier protein (e.g.,each capsular polysaccharide molecule being conjugated to a singlecarrier protein). In another embodiment, the capsular polysaccharidesnot conjugated to the first carrier protein are conjugated to two ormore carrier proteins (each capsular polysaccharide molecule beingconjugated to a single carrier protein). In such embodiments, eachcapsular polysaccharide of the same serotype is typically conjugated tothe same carrier protein.

Conjugation

Prior to conjugation, the purified polysaccharides can be chemicallyactivated to make the saccharides capable of reacting with the carrierprotein to form an activated polysaccharide. As used herein, the term“activated polysaccharide” refers to a polysaccharide that has beenchemically modified as described below to enable conjugation to a linkeror a carrier protein. The purified polysaccharides can optionally beconnected to a linker. Once activated or connected to a linker, eachcapsular polysaccharide is separately conjugated to a carrier protein toform a glycoconjugate. The polysaccharide conjugates may be prepared byknown coupling techniques.

In certain embodiments, the activation of S. pneumoniae serotype 23Apolysaccharide occurs on the α-Rhap or β-Glcp. In certain embodiments,the activation of S. pneumoniae serotype 23B polysaccharide occurs onthe β-Glcp or β-Rhap. In one aspect of this embodiment, activation ofserotype 23B polysaccharide occurs at greater than 90%, 95%, 99%, or100% on the β-Rhap. Surprisingly, despite the availability of a suitableactivation site on β-Glcp, activation occurs exclusively on the β-Rhap.In certain embodiments, the polysaccharide is activated with periodate.In certain aspects of this embodiment, the activation of serotype 23Apolysaccharide occurs on the 2^(nd) or 3^(rd) carbon position of α-Rhapor β-Glcp or the activation of serotype 23B polysaccharide occurs on the2^(nd) or 3^(rd) carbon position of β-Glcp or β-Rhap. In one sub-aspectof this aspect, the extent of activation of serotype 23B polysaccharideon the 2^(nd) or 3^(rd) carbon position of β-Rhap is greater than theextent of activation on the 2^(nd) or 3^(rd) carbon position of β-Glcp.The extent of activation on β-Rhap can be at least 60%, 70%, 80%, or90%. In another sub-aspect of this aspect, perodiate activation ofserotype 23B polysaccharide occurs at greater than 90%, 95%, 99% or 100%on the β-Rhap.

In certain embodiments, the polysaccharide can be coupled to a linker toform a polysaccharide-linker intermediate in which the free terminus ofthe linker is an ester group. The linker is therefore one in which atleast one terminus is an ester group. The other terminus is selected sothat it can react with the polysaccharide to form thepolysaccharide-linker intermediate.

In certain embodiments, the polysaccharide can be coupled to a linkerusing a primary amine group in the polysaccharide. In this case, thelinker typically has an ester group at both termini. This allows thecoupling to take place by reacting one of the ester groups with theprimary amine group in the polysaccharide by nucleophilic acylsubstitution. The reaction results in a polysaccharide-linkerintermediate in which the polysaccharide is coupled to the linker via anamide linkage. The linker is therefore a bifunctional linker thatprovides a first ester group for reacting with the primary amine groupin the polysaccharide and a second ester group for reacting with theprimary amine group in the carrier molecule. A typical linker is adipicacid N-hydroxysuccinimide diester (SIDEA).

In certain embodiments, the coupling can also take place indirectly,i.e. with an additional linker that is used to derivatise thepolysaccharide prior to coupling to the linker. The polysaccharide iscoupled to the additional linker using a carbonyl group at the reducingterminus of the polysaccharide. This coupling comprises two steps: (a1)reacting the carbonyl group with the additional linker; and (a2)reacting the free terminus of the additional linker with the linker. Inthese embodiments, the additional linker typically has a primary aminegroup at both termini, thereby allowing step (a1) to take place byreacting one of the primary amine groups with the carbonyl group in thepolysaccharide by reductive amination. A primary amine group is usedthat is reactive with the carbonyl group in the polysaccharide.Hydrazide or hydroxylamino groups are suitable. The same primary aminegroup is typically present at both termini of the additional linker. Thereaction results in a polysaccharide-additional linker intermediate inwhich the polysaccharide is coupled to the additional linker via a C—Nlinkage.

In certain embodiments, the polysaccharide can be coupled to theadditional linker using a different group in the polysaccharide,particularly a carboxyl group. This coupling comprises two steps: (a1)reacting the group with the additional linker; and (a2) reacting thefree terminus of the additional linker with the linker. In this case,the additional linker typically has a primary amine group at bothtermini, thereby allowing step (a1) to take place by reacting one of theprimary amine groups with the carboxyl group in the polysaccharide byEDAC activation. A primary amine group is used that is reactive with theEDAC-activated carboxyl group in the polysaccharide. A hydrazide groupis suitable. The same primary amine group is typically present at bothtermini of the additional linker. The reaction results in apolysaccharide-additional linker intermediate in which thepolysaccharide is coupled to the additional linker via an amide linkage.

In one embodiment, the chemical activation of the polysaccharides andsubsequent conjugation to the carrier protein by reductive amination canbe achieved by means described in U.S. Pat. Nos. 4,365,170, 4,673,574and 4,902,506, U.S. Patent Application Publication Nos. 2006/0228380,2007/184072, 2007/0231340 and 2007/0184071, and International PatentApplication Publication Nos. WO2006/110381, WO2008/079653, andWO2008/143709). The chemistry may entail the activation of pneumococcalpolysaccharide by reaction with any oxidizing agent which a primaryhydroxyl group to an aldehyde, such as TEMPO in the presence of oxidant(WO2104/097099), or reacting two vicinal hydroxyl groups to aldehydes,such as periodate (including sodium periodate, potassium periodate, orperiodic acid). The reactions lead to a random oxidation of primaryhydroxyl groups or random oxidative cleavage of vicinal hydroxyl groupsof the carbohydrates with the formation of reactive aldehyde groups.

In this embodiment, coupling to the carrier protein is by reductiveamination via direct amination to the lysyl groups of the protein. Forexample, conjugation is carried out by reacting a mixture of theactivated polysaccharide and carrier protein with a reducing agent suchas sodium cyanoborohydride in the presence of nickel. The conjugationreaction may take place under aqueous solution or in the presence ofdimethyl sulfoxide (DMSO). See, e.g., U.S. Patent ApplicationPublication Nos. US2015/0231270 and US2011/0195086 and European PatentNo. EP 0471 177 B1. Unreacted aldehydes are then capped with theaddition of a strong reducing agent, such as sodium borohydride.

Reductive amination involves two steps, (1) oxidation of thepolysaccharide to form reactive aldehydes, (2) reduction of the imine(Schiff base) formed between activated polysaccharide and a carrierprotein to form a stable amine conjugate bond. Before oxidation, thepolysaccharide is optionally size reduced. Mechanical methods (e.g.homogenization) or chemical hydrolysis may be employed. Chemicalhydrolysis may be conducted using acetic acid. The oxidation step mayinvolve reaction with periodate. For the purpose of the presentinvention, the term “periodate” includes both periodate and periodicacid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO₆ ⁵⁻) and includes the various salts of periodate(e.g., sodium periodate and potassium periodate). In an embodiment thecapsular polysaccharide is oxidized in the presence of metaperiodate,preferably in the presence of sodium periodate (NaIO₄). In anotherembodiment the capsular polysaccharide is oxydized in the presence oforthoperiodate, preferably in the presence of periodic acid.

In an embodiment, the oxidizing agent is a stable nitroxyl or nitroxideradical compound, such as piperidine-N-oxy or pyrrolidine-N-oxycompounds, in the presence of an oxidant to selectively oxidize primaryhydroxyls (as described in, for example, International PatentApplication Publication No. WO 2014/097099). In said reaction, theactual oxidant is the N-oxoammonium salt, in a catalytic cycle. In anaspect, said stable nitroxyl or nitroxide radical compound arepiperidine-N-oxy or pyrrolidine-N-oxy compounds. In an aspect, saidstable nitroxyl or nitroxide radical compound bears a TEMPO(2,2,6,6-tetramethyl-1-piperidinyloxy) or a PROXYL(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In an aspect, saidstable nitroxyl radical compound is TEMPO or a derivative thereof. In anaspect, said oxidant is a molecule bearing a N-halo moiety. In anaspect, said oxidant is selected from the group consisting ofN-ChloroSuccinimide, N-Bromosuccinimide, N-Iodosuccinimide,Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione,Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.Preferably said oxidant is N-Chlorosuccinimide.

In certain aspects, the oxidizing agent is2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) free radical andN-Chlorosuccinimide (NCS) as the cooxidant (as described inInternational Patent Application Publication No. WO2014/097099).Therefore in one aspect, the glycoconjugates from S. pneumoniae areobtainable by a method comprising the steps of: a) reacting a saccharidewith 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) andN-chlorosuccinimide (NCS) in an aqueous solvent to produce an activatedsaccharide; and b) reacting the activated saccharide with a carrierprotein comprising one or more amine groups (said method is designated“TEMPO/NCS-reductive amination” thereafter).

Optionally the oxidation reaction is quenched by addition of a quenchingagent. The quenching agent may be selected from vicinal diols,1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid (such as glycerol, ethylene glycol, propan-1,2-diol,butan-1,2-diol or butan-2,3-diol, ascorbic acid).

The second step of the conjugation process for reductive amination isthe reduction of the imine (Schiff base) bond between activatedpolysaccharide and a carrier protein to form a stable conjugate bond(so-called reductive amination), using a reducing agent. Reducing agentswhich are suitable include the cyanoborohydrides (such as sodiumcyanoborohydride) or sodium borohydride. In one embodiment the reducingagent is sodium cyanoborohydride.

In certain embodiments of the methods of the invention, the reductiveamination reaction is carried out in aprotic solvent (or a mixture ofaprotic solvents). In an embodiment, the reduction reaction is carriedout in DMSO (dimethyl sulfoxide) or in DMF (dimethylformamide) solvent.The DMSO or DMF solvent may be used to reconstitute the activatedpolysaccharide and carrier protein, if lyophilized. In one embodiment,the aprotic solvent is DMSO.

At the end of the reduction reaction, there may be unreacted aldehydegroups remaining in the conjugates, which may be capped or quenchedusing a suitable capping or quenching agent. In one embodiment thiscapping or quenching agent is sodium borohydride (NaBH₄). Suitablealternatives include sodium triacetoxyborohydride or sodium or zincborohydride in the presence of Bronsted or Lewis acids), amine boranessuch as pyridine borane, 2-Picoline Borane, 2,6-diborane-methanol,dimethylamine-borane, t-BuMe′PrN—BH₃, benzylamine-BH₃ or5-ethyl-2-methylpyridine borane (PEMB) or borohydride exchange resin.

Glycoconjugates prepared using reductive amination in an aprotic solventare generally used in multivalent pneumococcal conjugate vaccines. Thus,in certain embodiments for multivalent compositions where not all theserotypes are prepared in an aprotic solvent, the reduction reaction forthe remaining seroytpes is carried out in aqueous solvent (e.g.,selected from PBS (phosphate buffered saline), MES(2-(N-morpholino)ethanesulfonic acid), HEPES,(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-tris, ADA(N-(2-Acetamido)iminodiacetic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPSO(3-Morpholino-2-hydroxypropanesulfonic acid), BES(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), DIPSO (3-Bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS(4-(N-morpholino)butanesulfonic acid), HEPPSO(N-(2-Hydroxyethyl)piperazine-N-(2-hydroxypropanesulfonic acid)), POPSO(Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA(triethanolamine), EPPS (4-(2-Hydroxyethyl)piperazine-1-propanesulfonicacid), Bicine or HEPB, at a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0and 7.5).

In some embodiments, the glycoconjugates of the present inventioncomprise a polysaccharide having a molecular weight of between 10 kDaand 10,000 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 25 kDa and 5,000 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 50 kDaand 1,000 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 70 kDa and 900 kDa. In other suchembodiments, the polysaccharide has a molecular weight of between 100kDa and 800 kDa. In other such embodiments, the polysaccharide has amolecular weight of between 200 kDa and 600 kDa. In further suchembodiments, the polysaccharide has a molecular weight of 100 kDa to1,000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700 kDa;100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to 800 kDa;150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to400 kDa; 150 kDa to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900 kDa;200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDa to500 kDa; 200 kDa to 400 kDa; 200 kDa to 300; 250 kDa to 1,000 kDa; 250kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDa to 600kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDato 1,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to 700kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDato 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700kDa; 400 kDa to 600 kDa; or 500 kDa to 600 kDa.

In certain embodiments, the conjugation reaction is performed byreductive amination wherein nickel is used for greater conjugationreaction efficiency and to aid in free cyanide removal. Transitionmetals are known to form stable complexes with cyanide and are known toimprove reductive methylation of protein amino groups and formaldehydewith sodium cyanoborohydride (S Gidley et al., Biochem J. 1982, 203:331-334; Jentoft et al. Anal Biochem. 1980, 106: 186-190). By complexingresidual, inhibitory cyanide, the addition of nickel increases theconsumption of protein during the conjugation of and leads to formationof larger, potentially more immungenic conjugates.

Suitable alternative chemistries include the activation of thesaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester. The activated saccharide may thus becoupled directly or via a spacer (linker) group to an amino group on thecarrier protein. For example, the spacer could be cystamine orcysteamine to give a thiolated polysaccharide which could be coupled tothe carrier via a thioether linkage obtained after reaction with amaleimide-activated carrier protein (for example using GMBS) or ahaloacetylated carrier protein (for example using iodoacetimide [e.g.ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB, or SIA,or SBAP). Preferably, the cyanate ester (optionally made by CDAPchemistry) is coupled with hexane diamine or adipic acid dihydrazide(ADH) and the amino-derivatised saccharide is conjugated to the carrierprotein using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxylgroup on the protein carrier. Such conjugates are described inInternational Patent Application Publication Nos. WO 93/15760, WO95/08348 and WO 96/29094; and Chu et al., 1983, Infect. Immunity40:245-256.

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S—NHS, EDC, TSTU.Many are described in International Patent Application Publication No.WO 98/42721. Conjugation may involve a carbonyl linker which may beformed by reaction of a free hydroxyl group of the saccharide with CDI(See Bethell et al., 1979, J. Biol. Chem. 254:2572-4; Hearn et al.,1981, J. Chromatogr. 218:509-18) followed by reaction with a protein toform a carbamate linkage. This may involve reduction of the anomericterminus to a primary hydroxyl group, optional protection/deprotectionof the primary hydroxyl group, reaction of the primary hydroxyl groupwith CDI to form a CDI carbamate intermediate and coupling the CDIcarbamate intermediate with an amino group on a protein.

Following the conjugation (the reduction reaction and optionally thecapping or quenching reaction), the glycoconjugates may be purified(enriched with respect to the amount of polysaccharide-proteinconjugate) by a variety of techniques known to the skilled person. Thesetechniques include dialysis, concentration/diafiltration operations,tangential flow filtration, ultrafiltration, precipitation/elution,column chromatography (ion exchange chromatography, multimodal ionexchange chromatography, DEAE, or hydrophobic interactionchromatography), and depth filtration. See, e.g., U.S. Pat. No.6,146,902. In an embodiment, the glycoconjugates are purified bydiafilitration or ion exchange chromatography or size exclusionchromatography.

One way to characterize the glycoconjugates of the invention is by thenumber of lysine residues in the carrier protein (e.g., CRM197) thatbecome conjugated to the saccharide, which can be characterized as arange of conjugated lysines (degree of conjugation). The evidence forlysine modification of the carrier protein, due to covalent linkages tothe polysaccharides, can be obtained by amino acid analysis usingroutine methods known to those of skill in the art. Conjugation resultsin a reduction in the number of lysine residues recovered, compared tothe carrier protein starting material used to generate the conjugatematerials. In a preferred embodiment, the degree of conjugation of theglycoconjugate of the invention is between 2 and 15, between 2 and 13,between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5,between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10,between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4,between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12,between 10 and 15 or between 10 and 12. In an embodiment, the degree ofconjugation of the glycoconjugate of the invention is about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14 or about 15. In a preferred embodiment,the degree of conjugation of the glycoconjugate of the invention isbetween 4 and 7. In some such embodiments, the carrier protein isCRM197.

The glycoconjugates of the invention may also be characterized by theratio (weight/weight) of saccharide to carrier protein. In someembodiments, the ratio of polysaccharide to carrier protein in theglycoconjugate (w/w) is between 0.5 and 3.0 (e.g., about 0.5, about 0.6,about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9,about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In otherembodiments, the saccharide to carrier protein ratio (w/w) is between0.5 and 2.0, between 0.5 and 1.5, between 0.8 and 1.2, between 0.5 and1.0, between 1.0 and 1.5 or between 1.0 and 2.0. In further embodiments,the saccharide to carrier protein ratio (w/w) is between 0.8 and 1.2. Ina preferred embodiment, the ratio of capsular polysaccharide to carrierprotein in the conjugate is between 0.9 and 1.1. In some suchembodiments, the carrier protein is CRM197. The glycoconjugates andimmunogenic compositions of the invention may contain free saccharidethat is not covalently conjugated to the carrier protein, but isnevertheless present in the glycoconjugate composition. The freesaccharide may be non-covalently associated with (i.e., non-covalentlybound to, adsorbed to, or entrapped in or with) the glycoconjugate.

In a preferred embodiment, the glycoconjugate comprises less than about50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of free polysaccharide comparedto the total amount of polysaccharide. In a preferred embodiment theglycoconjugate comprises less than about 25% of free polysaccharidecompared to the total amount of polysaccharide. In a preferredembodiment the glycoconjugate comprises less than about 20% of freepolysaccharide compared to the total amount of polysaccharide. In apreferred embodiment the glycoconjugate comprises less than about 15% offree polysaccharide compared to the total amount of polysaccharide.

Multivalent Polysaccharide-Protein Conjugate Vaccines

In certain embodiments of the invention, multivalent polysaccharidevaccines comprise unconjugated polysaccharides or polysaccharide-proteinconjugates from one or more of Streptococcus pneumoniae serotypes 23Aand 23B and capsular polysaccharides from one or more of S. pneumoniaeserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A,11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18B, 18C, 19A, 19F, 20, 21, 22A,22F, 23F, 24B, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F, and 38 eitheras free polysaccharides, a component of a polysaccharide-proteinconjugate or a combination thereof, to provide a multivalentpneumococcal vaccine. In certain embodiments of the invention, theimmunogenic composition comprises, consists essentially of, or consistsof capsular polysaccharides from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 S. pneumoniaeserotypes individually conjugated to one or more carrier proteins.Preferably, saccharides from a particular serotype are not conjugated tomore than one carrier protein.

After the individual glycoconjugates are purified, they are compoundedto formulate the immunogenic composition of the present invention. Thesepneumococcal conjugates are prepared by separate processes and bulkformulated into a single dosage formulation.

For certain serotypes within S. pneumoniae serogroup 23,cross-protection can be seen. In other words, polysaccharides from aparticular serotype may induce an immune response which is protectiveagainst another serotype. Typically, the other serotype is within thesame serogroup, but in some cases, one serotype may provide protectionto a serotype in a different serogroup. On the other hand, sometimescross-protection is not seen even in the same serogroup withpolysaccharides from these serotypes having similar structures. TheEXAMPLES unexpectedly demonstrate that polysaccharides from S.pneumoniae serotypes 23A and 23F offer weak cross-protection against S.pneumoniae serotype 23B (while providing robust cross protection againsteach other). Thus, in order to obtain protection against serotypes 23A,23B and 23F, a multivalent composition comprising polysaccharideserotypes 23A or 23F needs to include serotype 23B polysaccharide.Accordingly, in certain embodiments of the invention, a multivalentcomposition comprises polysaccharides from serotypes 23A and 23B. Inother embodiments of the invention, a multivalent composition comprisespolysaccharides from serotypes 23F and 23B.

Pharmaceutical/Vaccine Compositions

The present invention further provides compositions, includingpharmaceutical, immunogenic and vaccine compositions, comprising,consisting essentially of, or alternatively, consisting of any of thepolysaccharide S. pneumoniae serotype combinations described abovetogether with a pharmaceutically acceptable carrier and an adjuvant.

Formulation of the polysaccharide-protein conjugates of the presentinvention can be accomplished using art-recognized methods. Forinstance, individual pneumococcal conjugates can be formulated with aphysiologically acceptable vehicle to prepare the composition. Examplesof such vehicles include, but are not limited to, water, bufferedsaline, polyols (e.g., glycerol, propylene glycol, liquid polyethyleneglycol) and dextrose solutions.

In a preferred embodiment, the vaccine composition is formulated inL-histidine buffer with sodium chloride.

As defined herein, an “adjuvant” is a substance that serves to enhancethe immunogenicity of an immunogenic composition of the invention. Animmune adjuvant may enhance an immune response to an antigen that isweakly immunogenic when administered alone, e.g., inducing no or weakantibody titers or cell-mediated immune response, increase antibodytiters to the antigen, and/or lowers the dose of the antigen effectiveto achieve an immune response in the individual. Thus, adjuvants areoften given to boost the immune response and are well known to theskilled artisan. Suitable adjuvants to enhance effectiveness of thecomposition include, but are not limited to:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example, (a) MF59(International Patent Application Publication No. WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, (c) Ribi™ adjuvant system (RAS), (Corixa,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting of3-O-deacylated monophosphorylipid A (MPL™) described in U.S. Pat. No.4,912,094, trehalose dimycolate (TDM), and cell wall skeleton (CWS),preferably MPL+CWS (Detox™); and (d) a Montanide ISA;

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, Mass.) (see, e.g., U.S. Pat. No. 5,057,540) may be used orparticles generated therefrom such as ISCOM (immunostimulating complexesformed by the combination of cholesterol, saponin, phospholipid, andamphipathic proteins) and Iscomatrix® (having essentially the samestructure as an ISCOM but without the protein);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside,which is also known as 529 (formerly known as RC529), which isformulated as an aqueous form or as a stable emulsion;

(5) synthetic polynucleotides such as oligonucleotides containing CpGmotif(s) (U.S. Pat. No. 6,207,646);

(6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF),costimulatory molecules B7-1 and B7-2, etc; and

(7) complement, such as a trimer of complement component C3d.

In another embodiment, the adjuvant is a mixture of 2, 3, or more of theabove adjuvants, e.g., SBAS2 (an oil-in-water emulsion also containing3-deacylated monophosphoryl lipid A and QS21).

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′,2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

In certain embodiments, the adjuvant is an aluminum salt. The aluminumsalt adjuvant may be an alum-precipitated vaccine or an alum-adsorbedvaccine. Aluminum-salt adjuvants are well known in the art and aredescribed, for example, in Harlow, E. and D. Lane (1988; Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992;Aluminum salts. Research in Immunology 143:489-493). The aluminum saltincludes, but is not limited to, hydrated alumina, alumina hydrate,alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate,Alhydrogel®, Superfos, Amphogel®, aluminum (III) hydroxide, aluminumhydroxyphosphate (Aluminum Phosphate Adjuvant (APA)), amorphous alumina,trihydrated alumina, or trihydroxyaluminum.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA ismanufactured by blending aluminum chloride and sodium phosphate in a 1:1volumetric ratio to precipitate aluminum hydroxyphosphate. After theblending process, the material is size-reduced with a high-shear mixerto achieve a monodisperse particle size distribution. The product isthen diafiltered against physiological saline and steam sterilized.

In certain embodiments, a commercially available Al(OH)₃ (e.g.Alhydrogel® or Superfos of Denmark/Accurate Chemical and Scientific Co.,Westbury, N.Y.) is used to adsorb proteins. Adsorption of protein isdependent, in another embodiment, on the pI (Isoelectric pH) of theprotein and the pH of the medium. A protein with a lower pI adsorbs tothe positively charged aluminum ion more strongly than a protein with ahigher pI. Aluminum salts may establish a depot of Ag that is releasedslowly over a period of 2-3 weeks, be involved in nonspecific activationof macrophages and complement activation, and/or stimulate innate immunemechanism (possibly through stimulation of uric acid). See, e.g.,Lambrecht et al., 2009, Curr Opin Immunol 21:23.

Monovalent bulk aqueous conjugates are typically blended together anddiluted. Once diluted, the batch is sterile filtered. Aluminum phosphateadjuvant is added aseptically to target a final concentration of 4 μg/mLfor all S. pneumoniae serotypes except serotype 6B, which is diluted toa target of 8 μg/mL, and a final aluminum concentration of 250 μg/mL.The adjuvanted, formulated batch will be filled into vials or syringes.

In certain embodiments, the adjuvant is a CpG-containing nucleotidesequence, for example, a CpG-containing oligonucleotide, in particular,a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment,the adjuvant is ODN 1826, which may be acquired from ColeyPharmaceutical Group.

“CpG-containing nucleotide,” “CpG-containing oligonucleotide,” “CpGoligonucleotide,” and similar terms refer to a nucleotide molecule of6-50 nucleotides in length that contains an unmethylated CpG moiety.See, e.g., Wang et al., 2003, Vaccine 21:4297. In another embodiment,any other art-accepted definition of the terms is intended.CpG-containing oligonucleotides include modified oligonucleotides usingany synthetic internucleoside linkages, modified base and/or modifiedsugar.

Methods for use of CpG oligonucleotides are well known in the art andare described, for example, in Sur et al., 1999, J Immunol. 162:6284-93;Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006,Crit Rev Ther Drug Carrier Syst. 23:89-110.

Administration/Dosage

The compositions and formulations of the present invention can be usedto protect or treat a human susceptible to infection, e.g., apneumococcal infection, by means of administering the vaccine via asystemic or mucosal route. In one embodiment, the present inventionprovides a method of inducing an immune response to a S. pneumoniaecapsular polysaccharide conjugate, comprising administering to a humanan immunologically effective amount of an immunogenic composition of thepresent invention. In another embodiment, the present invention providesa method of vaccinating a human against a pneumococcal infection,comprising the step of administering to the human an immunogicallyeffective amount of an immunogenic composition of the present invention.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, in another embodiment, thedosage for human vaccination is determined by extrapolation from animalstudies to human data. In another embodiment, the dosage is determinedempirically.

“Effective amount” of a composition of the invention refers to a doserequired to elicit antibodies that significantly reduce the likelihoodor severity of infectivitiy of a microbe, e.g., S. pneumoniae, during asubsequent challenge.

The methods of the invention can be used for the prevention and/orreduction of primary clinical syndromes caused by microbes, e.g., S.pneumoniae, including both invasive infections (meningitis, pneumonia,and bacteremia), and noninvasive infections (acute otitis media, andsinusitis).

Administration of the compositions of the invention can include one ormore of: injection via the intramuscular, intraperitoneal, intradermalor subcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory or genitourinary tracts. In one embodiment,intranasal administration is used for the treatment of pneumonia orotitis media (as nasopharyngeal carriage of pneumococci can be moreeffectively prevented, thus attenuating infection at its earlieststage).

The amount of conjugate in each vaccine dose is selected as an amountthat induces an immunoprotective response without significant, adverseeffects. Such amount can vary depending upon the pneumococcal serotype.Generally, for polysaccharide-based conjugates, each dose will comprise0.1 to 100 μg of each polysaccharide, particularly 0.1 to 10 μg, andmore particularly 1 to 5 μg. For example, each dose can comprise 100,150, 200, 250, 300, 400, 500, or 750 ng or 1, 1.5, 2, 3, 4, 5, 6, 7,7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60,70, 80, 90, or 100 μg of each polysaccharide.

Optimal amounts of components for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. For example, in another embodiment, thedosage for human vaccination is determined by extrapolation from animalstudies to human data. In another embodiment, the dosage is determinedempirically.

In one embodiment, the dose of the aluminum salt is 10, 15, 20, 25, 30,50, 70, 100, 125, 150, 200, 300, 500, or 700 μg, or 1, 1.2, 1.5, 2, 3, 5mg or more. In yet another embodiment, the dose of aluminum saltdescribed above is per μg of recombinant protein.

Generally, each 0.5 mL dose is formulated to contain: 2 μg of each S.pneumoniae polysaccharide, except for serotype 6B polysaccharide at 4μg; about 32 μg CRM197 carrier protein (e.g., 32 μg±5 μg, ±3 μg, ±2 μg,or ±1 μg); 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate)adjuvant; and sodium chloride and L-histidine buffer. The sodiumchloride concentration is about 150 mM (e.g., 150 mM±25 mM, ±20 mM, ±15mM, ±10 mM, or ±5 mM) and about 20 mM (e.g., 20 mM±5 mM, ±2.5 mM, ±2 mM,±1 mM, or ±0.5 mM) L-histidine buffer.

According to any of the methods of the present invention and in oneembodiment, the subject is human. In certain embodiments, the humanpatient is an infant (less than 1 year of age), toddler (approximately12 to 24 months), or young child (approximately 2 to 5 years). In otherembodiments, the human patient is an elderly patient (>65 years). Thecompositions of this invention are also suitable for use with olderchildren, adolescents and adults (e.g., aged 18 to 45 years or 18 to 65years).

In one embodiment of the methods of the present invention, a compositionof the present invention is administered as a single inoculation. Inanother embodiment, the composition is administered twice, three timesor four times or more, adequately spaced apart. For example, thecomposition may be administered at 1, 2, 3, 4, 5, or 6 month intervalsor any combination thereof. The immunization schedule can follow thatdesignated for pneumococcal vaccines. For example, the routine schedulefor infants and toddlers against invasive disease caused by S.pneumoniae is 2, 4, 6 and 12-15 months of age. Thus, in a preferredembodiment, the composition is administered as a 4-dose series at 2, 4,6, and 12-15 months of age.

The compositions of this invention may also include one or more proteinsfrom S. pneumoniae. Examples of S. pneumoniae proteins suitable forinclusion include those identified in International Patent ApplicationPublication Nos. WO 02/083855 and WO 02/053761.

Formulations

The compositions of the invention can be administered to a subject byone or more method known to a person skilled in the art, such asparenterally, transmucosally, transdermally, intramuscularly,intravenously, intra-dermally, intra-nasally, subcutaneously,intra-peritonealy, and formulated accordingly.

In one embodiment, compositions of the present invention areadministered via epidermal injection, intramuscular injection,intravenous, intra-arterial, subcutaneous injection, orintra-respiratory mucosal injection of a liquid preparation. Liquidformulations for injection include solutions and the like.

The composition of the invention can be formulated as single dose vials,multi-dose vials or as pre-filled syringes.

In another embodiment, compositions of the present invention areadministered orally, and are thus formulated in a form suitable for oraladministration, i.e., as a solid or a liquid preparation. Solid oralformulations include tablets, capsules, pills, granules, pellets and thelike. Liquid oral formulations include solutions, suspensions,dispersions, emulsions, oils and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueousor non-aqueous solutions, suspensions, emulsions or oils. Examples ofnonaqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Examples of oils are those of animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipidfrom milk or eggs.

The pharmaceutical composition may be isotonic, hypotonic or hypertonic.However, it is often preferred that a pharmaceutical composition forinfusion or injection is essentially isotonic when it is administrated.Hence, for storage the pharmaceutical composition may preferably beisotonic or hypertonic. If the pharmaceutical composition is hypertonicfor storage, it may be diluted to become an isotonic solution prior toadministration.

The isotonic agent may be an ionic isotonic agent such as a salt or anon-ionic isotonic agent such as a carbohydrate. Examples of ionicisotonic agents include but are not limited to NaCl, CaCl₂, KCl andMgCl₂. Examples of non-ionic isotonic agents include but are not limitedto sucrose, trehalose, mannitol, sorbitol and glycerol.

It is also preferred that at least one pharmaceutically acceptableadditive is a buffer. For some purposes, for example, when thepharmaceutical composition is meant for infusion or injection, it isoften desirable that the composition comprises a buffer, which iscapable of buffering a solution to a pH in the range of 4 to 10, such as5 to 9, for example 6 to 8.

The buffer may for example be selected from the group consisting ofTris, acetate, glutamate, lactate, maleate, tartrate, phosphate,citrate, carbonate, glycinate, L-histidine, glycine, succinate andtriethanolamine buffer.

The buffer may furthermore for example be selected from USP compatiblebuffers for parenteral use, in particular, when the pharmaceuticalformulation is for parenteral use. For example the buffer may beselected from the group consisting of monobasic acids such as acetic,benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic,adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric,polybasic acids such as citric and phosphoric; and bases such asammonia, diethanolamine, glycine, triethanolamine, and Tris.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, glycols such as propylene glycols orpolyethylene glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), andPoloxamer 188 (P188) are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, olive oil,sunflower oil, fish-liver oil, another marine oil, or a lipid from milkor eggs.

The formulations may also contain a surfactant. Preferred surfactantsinclude, but are not limited to: the polyoxyethylene sorbitan esterssurfactants (commonly referred to as the Tweens), especially PS-20 andPS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/orbutylene oxide (BO), sold under the DOWFAX™ tradename, such as linearEO/PO block copolymers; octoxynols, which can vary in the number ofrepeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (TritonX-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. A preferred surfactant for including inthe emulsion is PS-20 or PS-80.

Mixtures of surfactants can be used, e.g. PS-80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (PS-80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants are: polyoxyethylene sorbitan esters(such as PS-80) 0.01 to 1% w/v, in particular about 0.1% w/v; octyl- ornonylphenoxy polyoxyethanols (such as Triton X-100, or other detergentsin the Triton series) 0.001 to 0.1% w/v, in particular 0.005 to 0.02%w/v; polyoxyethylene ethers (such as laureth 9) 0.1 to 20% w/v,preferably 0.1 to 10% w/v and in particular 0.1 to 1% w/v or about 0.5%w/v.

In certain embodiments, the composition consists essentially ofL-histidine (20 mM), saline (150 mM) and 0.2% w/v PS-20 at a pH of 5.8with 250 μg/mL of APA (Aluminum Phosphate Adjuvant). PS-20 can rangefrom 0.005 to 0.1% w/v with the presence of PS-20 or PS-80 informulation controlling aggregation during simulated manufacture and inshipping using primary packaging. Process consists of combining blend ofup to 44 S. pneumoniae polysaccharide serotypes in L-histidine, sodiumchloride, and PS-20 then combining this blended material with APA andsodium chloride with or without antimicrobial preservatives.

The choice of surfactant may need to be optimized for different drugproducts and drug substances. For multivalent vaccines containing 15 ormore S. pneumoniae polysaccharide serotypes, PS-20 and P188 arepreferred. The choice of chemistry used to prepare the conjugate canalso influence the stabilization of the formulation. In particular, asexemplified below, pneumococcal polysaccharide-protein conjugatesprepared in aqueous or DMSO solvent and combined in a multivalentcomposition show significant differences in stability depending on theparticular surfactant systems used for formulation.

For the formulations described herein, a poloxamer generally has amolecular weight in the range from 1,100 Da to 17,400 Da, from 7,500 Dato 15,000 Da, or from 7,500 Da to 10,000 Da. The poloxamer can beselected from poloxamer 188 or poloxamer 407. The final concentration ofthe poloxamer in the formulations of the invention is from 0.001 to 5%w/v, or 0.025 to 1% w/v. A surfactant system comprising a poloxamer mustfurther comprise a polyol. In certain aspects, the polyol is propyleneglycol and is at final concentration from 1 to 20% w/v. In certainaspects, the polyol is polyethylene glycol 400 and is at finalconcentration from 1 to 20% w/v.

Suitable polyols for the formulations are polymeric polyols,particularly polyether diols including, but are not limited to,propylene glycol and polyethylene glycol, Polyethylene glycol monomethylethers. Propylene glycol is available in a range of molecular weights ofthe monomer from ˜425 Da to ˜2,700 Da. Polyethylene glycol andPolyethylene glycol monomethyl ether is also available in a range ofmolecular weights ranging from ˜200 Da to ˜35,000 Da including but notlimited to PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600,PEG MME 2000, PEG MME 3350 and PEG MME 4000. A preferred polyethyleneglycol is polyethylene glycol 400. The final concentration of the polyolin the formulations may be 1 to 20% w/v or 6 to 20% w/v.

The formulation also contains a pH-buffered saline solution. The buffermay, for example, be selected from the group consisting of Tris,acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate,carbonate, glycinate, L-histidine, glycine, succinate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. Thebuffer is capable of buffering a solution to a pH in the range of 4 to10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspects, the buffer selectedfrom the group consisting of phosphate, succinate, L-histidine, MES,MOPS, HEPES, acetate or citrate. The buffer may furthermore, forexample, be selected from USP compatible buffers for parenteral use, inparticular, when the pharmaceutical formulation is for parenteral use.The concentrations of buffer will range from 1 mM to 50 mM or 5 mM to 50mM. In certain aspects, the buffer is L-histidine at a finalconcentration of 5 mM to 50 mM, or succinate at a final concentration of1 mM to 10 mM. In certain aspects, the L-histidine is at a finalconcentration of 20 mM±2 mM.

While the saline solution (i.e., a solution containing NaCl) ispreferred, other salts suitable for formulation include but are notlimited to, CaCl₂, KCl and MgCl₂ and combinations thereof. Non-ionicisotonic agents including but not limited to sucrose, trehalose,mannitol, sorbitol and glycerol may be used in lieu of a salt. Suitablesalt ranges include, but not are limited to 25 mM to 500 mM or 40 mM to170 mM. In one aspect, the saline is NaCl, optionally present at aconcentration from 20 mM to 170 mM.

In a preferred embodiment, the formulations comprise a L-histidinebuffer with sodium chloride.

In another embodiment, the pharmaceutical composition is delivered in acontrolled release system. For example, the agent can be administeredusing intravenous infusion, a transdermal patch, liposomes, or othermodes of administration. In another embodiment, polymeric materials areused; e.g. in microspheres in or an implant.

The compositions of this invention may also include one or more proteinsfrom S. pneumoniae. Examples of S. pneumoniae proteins suitable forinclusion include those identified in International Patent ApplicationPublication Nos. WO 02/083855 and WO 02/053761.

Analytical Methods

Molecular Weight and Concentration Analysis of Conjugates UsingHPSEC/UV/MALS/RI Assay

Conjugate samples are injected and separated by high performancesize-exclusion chromatography (HPSEC). Detection is accomplished withultraviolet (UV), multi-angle light scattering (MALS) and refractiveindex (RI) detectors in series. Protein concentration is calculated fromUV280 using an extinction coefficient. Polysaccharide concentration isdeconvoluted from the RI signal (contributed by both protein andpolysaccharide) using the do/dc factors which are the change in asolution's refractive index with a change in the solute concentrationreported in mL/g. Average molecular weight of the samples are calculatedby Astra software (Wyatt Technology Corporation, Santa Barbara, Calif.)using the measured concentration and light scattering information acrossthe entire sample peak. There are multiple forms of average values ofmolecular weight for polydispersed molecules. For example,number-average molecular weight Mn, weight-average molecular weight Mw,and z-average molecular weight Mz (Molecules, 2015, 20:10313-10341).Unless specified, the term “molecular weight”, as used throughout thespecification, is the weight-average molecular weight.

Determination of Lysine Consumption in Conjugated Protein as a Measureof the Number of Covalent Attachments Between Polysaccharide and CarrierProtein

The Waters AccQ-Tag amino acid analysis (AAA) is used to measure theextent of conjugation in conjugate samples. Samples are hydrolyzed usingvapor phase acid hydrolysis in the Eldex workstation, to break thecarrier proteins down into their component amino acids. The free aminoacids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC). The derivatized samples are then analyzed using UPLCwith UV detection on a C18 column. The average protein concentration isobtained using representative amino acids other than lysine. Lysineconsumption during conjugation (i.e., lysine loss) is determined by thedifference between the average measured amount of lysine in theconjugate and the expected amount of lysine in the starting protein.

Free Polysaccharide Testing

Free polysaccharide (i.e., polysaccharide that is not conjugated withCRM197) in the conjugate sample is measured by first precipitating freeprotein and conjugates with deoxycholate (DOC) and hydrochloric acid.Precipitates are then filtered out and the filtrates are analyzed forfree polysaccharide concentration by HPSEC/UV/MALS/RI. Freepolysaccharide is calculated as a percentage of total polysaccharidemeasured by HPSEC/UV/MALS/RI.

Free Protein Testing

Free polysaccharide, polysaccharide-CRM197 conjugate, and free CRM197 inthe conjugate samples are separated by capillary electrophoresis inmicellar electrokinetic chromatography (MEKC) mode. Briefly, samples aremixed with MEKC running buffer containing 25 mM borate, 100 mM SDS, pH9.3, and are separated in a preconditioned bare-fused silica capillary.Separation is monitored at 200 nm and free CRM197 is quantified with aCRM197 standard curve. Free protein results are reported as a percentageof total protein content determined by the HPSEC/UV/MALS/RI procedure.

Having described various embodiments of the invention with reference tothe accompanying description and drawings, it is to be understood thatthe invention is not limited to those precise embodiments, and thatvarious changes and modifications may be effected therein by one skilledin the art without departing from the scope or spirit of the inventionas defined in the appended claims.

The following examples illustrate but do not limit the invention.

EXAMPLES Example 1 Preparation of S. Pneumoniae Capsular Polysaccharides

Methods of culturing pneumococci are well known in the art. See, e.g.,Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods ofpreparing pneumococcal capsular polysaccharides are also well known inthe art. See, e.g., European Patent No. EP 0 497 524 B1. The processdescribed below generally follows the method described in EuropeanPatent No. EP 0 497 524 B1 and is generally applicable to allpneumococcal serotypes except where specifically modified.

Isolates of pneumococcal subtypes 23A and 23F were obtained from theMerck Culture Collection. Strain for serotype 23B was obtained fromCenters for Disease Control and Prevention (Atlanta, Ga.). Where needed,subtypes can be differentiated on the basis of Quelling reaction usingspecific antisera. See, e.g., U.S. Pat. No. 5,847,112. The obtainedisolates were further clonally isolated by plating serially in twostages on agar plates consisting of an animal-component free mediumcontaining soy peptone, yeast extract, and glucose without hemin (exceptfor serotype 23F, which contained hemin). Clonal isolates for eachserotype were further expanded in liquid culture using animal-componentfree media containing soy peptone, yeast extract, HEPES, sodiumchloride, sodium bicarbonate, potassium phosphate, glucose, and glycerolto prepare the pre-master cell banks.

The production of each serotype of pneumococcal polysaccharide consistedof a cell expansion and batch production fermentation followed bychemical inactivation prior to downstream purification. For serotypesother than 23F, a thawed cell bank vial from each serotype was expandedusing a shake flask or culture bottle containing a pre-sterilizedanimal-component free growth media containing soy peptone or soy peptoneultrafiltrate, yeast extract or yeast extract ultrafiltrate, HEPES,sodium chloride, sodium bicarbonate, potassium phosphate, and glucose.The cell expansion culture was grown in a sealed shake flask or bottleto minimize gas exchange with temperature and agitation control. Forserotype 23F, a thawed calle bank vial was expanded using a fermentorcontaining the same media. During the cell expansion of serotype 23F,temperature, pH, pressure, and agitation were controlled. Airflowoverlay was also controlled as sparging was not used.

After achieving a specified culture density, as measured by opticaldensity at 600 nm, a portion of the cell expansion culture wastransferred to a production fermentor containing pre-sterilizedanimal-component free growth media containing soy peptone or soy peptoneultrafiltrate, yeast extract or yeast extract ultrafiltrate, sodiumchloride, potassium phosphate, and glucose. Temperature, pH, pressure,and agitation were controlled. Airflow overlay was also controlled assparging was not used.

The batch fermentation was terminated via the addition of a chemicalinactivating agent, phenol, when glucose was nearly exhausted. Purephenol was added to a final concentration of 0.8-1.2% to inactivate thecells and liberate the capsular polysaccharide from the cell wall.Primary inactivation occurs for a specified time within the fermentorwhere temperature and agitation continue are to be controlled. Afterprimary inactivation, the batch was transferred to another vessel whereit was held for an additional specified time at controlled temperatureand agitation for complete inactivation. This was confirmed by eithermicrobial plating techniques or by verification of the phenolconcentration and specified time. The inactivated broth was thenpurified.

Purification of Ps

The purification of the pneumococcal polysaccharide consisted of severalcentrifugation, depth filtration, concentration/diafiltrationoperations, and precipitation steps. All procedures were performed atroom temperature unless otherwise specified.

Inactivated broth from the fermentor cultures of S. pneumoniae wereflocculated with a cationic polymer (such as BPA-1000, Petrolite“Tretolite” and “Spectrum 8160” and poly(ethyleneimine), “MilliporepDADMAC”). The cationic polymers binded to the impurity protein, nucleicacids and cell debris. Following the flocculation step and an agingperiod, flocculated solids were removed via centrifugation and multipledepth filtration steps. Clarified broth was concentrated and diafilteredusing a 100 kDa to 500 kDa MWCO (molecular weight cutoff) filter.Diafiltration was accomplished using Tris, MgCl₂ buffer and sodiumphosphate buffer. Diafiltration removed residual nucleic acid andprotein.

Further impurities removal was accomplished by reprecipitation of thepolysaccharide in sodium acetate and phenol with denatured alcoholand/or isopropanol. During the phenol precipitation step, sodium acetatein sodium phosphate saline buffer and phenol (liquefied phenols or solidphenols) was charged to the diafiltered retentate. Alcohol fractionationof the polysaccharide was then conducted in two stages. In the firststage a low percent alcohol was added to the preparation to precipitatecellular debris and other unwanted impurities, while the crudepolysaccharide remained in solution. The impurities were removed viacentrifugation followed by a depth filtration step. The polysaccharidewas then recovered from the solution by adding additional isopropanol ordenatured alcohol to the batch. The precipitated polysaccharide pelletwas recovered by centrifugation, triturated and dried as a powder andstored frozen at −70° C.

Example 2 NMR Structure Analyses of Polysaccharides

The strategy for determining polysaccharide structure involved amultiple step process performed substantially as described inAbeygunawardana et al., Determination of the Chemical Structure ofComplex Polysaccharides by Heteronuclear NMR Spectroscopy in Advances inBiophysical Chemistry 1993, Vol 3, pages 199-249, JAI Press Inc. Thepurified polysaccharides were examined using standard 1D and 2D NMRtechniques. Finally, the polysaccharides were examined for the presenceof phosphate using ³¹P NMR.

Assignments of the monosaccharide residues were carried out through¹H-¹H COSY, double quantum filtered homonuclear COSY and totalcorrelation spectroscopy (TOCSY). ¹³C chemical shifts were assigned byheteronuclear single quantum coherence spectroscopy (HSQC) andcombination HSQC-TOCSY. Multiplicity-edited HSQC was used to distinguishmethylene from methine groups. Inter-residue linkages were determinedthrough a combination of HMBC and NOESY spectroscopy. The anomericconfiguration of the residues was determined from the anomeric protonand carbon chemical shifts, ³J_(H1,H2) and ¹J_(H1,C1) values.

1D Phosphorus NMR spectroscopy indicated S. pneumoniae serotypes 23A and23B polysaccharides contained phosphorus in the structure. Assignment ofthe phosphorus linkage site was determined through ¹H-³¹P HMBC.

Based on the NMR data in FIGS. 2-5, the structure for S. pneumoniaeserotype 23A polysaccharide was determined to be as follows:

wherein n represents the number of repeating units constituting thepolysaccharide. See also FIG. 1A.

Based on the NMR data in FIGS. 2-5, the structure for S. pneumoniaeserotype 23B polysaccharide was determined to be as follows:

wherein n represents the number of repeating units constituting thepolysaccharide. See also FIG. 1B.

The sugar residues for S. pneumoniae serotype 23A and 23Bpolysaccharides include rhamnose (Rha), galactose (Gal), glucose (Glc)and glycerol.

The italicized letters (p and f) refer to pyranose (a closed ringconsisting of six atoms) and furanose (a closed ring consisting of fiveatoms).

The α and β refer to the configuration of the proton attached toanomeric carbon of the sugar unit. The anomeric carbon is always number1 when labeling the carbon atom in a sugar unit (usually 1 through 6). αmeans the anomeric proton is in the equatorial position in the 3Dstructure. β means the anomeric proton is in the axial position.

The numbers associated with arrows refer to how the individual sugarunits are connected to each other. For example, the nomenclatureα-Rhap-(1→3)-α-Glcp- means the number 1 carbon of Rhamnose is linked tothe number 3 carbon of Glucose (p means they are both pyranose rings).

Identification of Activation Sites

Activation sites were identified by reacting the aldehydes (usuallyhydrated) with thiosemicarbazide (TSC) in 5 mM citrate buffer. TSCreacts with aldehydes (as well as hydrated aldehydes) to form an imine(secondary aldimine). The imine proton formed has a unique chemicalshift that is downfield of the polysaccharide signals and was used toprobe the oxidation sites of the polysaccharide.

Oxidized S. pneumoniae serotype 23B polysaccharide was diluted withsodium citrate buffer then reacted with thiosemicarbazide, mixedcontinuously at ambient temperature, and then lyophilized. Thelyophilized sample was dissolved with 0.9 mL deuterium oxide for NMRanalysis.

NMR experiments were carried out at 600 MHz at probe temperature of 25°C. using a cryogenically cooled probe. A 1D proton spectrum was acquiredusing a 90 degree pulse with 16 transients and a 10 second delay betweenpulses (including 3 seconds of acquisition time). TOCSY and GradientCOSY data were acquired with 4 transients in the first dimension and 256and 512 increments in the second dimension respectively. NOESY data wereacquired with 16 transients in the first dimension and 256 increments inthe second dimension.

After TSC derivatization, all activated aldehydes were transformed toimine. The chemical shift of aldehyde protons were moved to 7-8 ppm,FIG. 6. The experiment result suggested two group of peaks are formedbetween 7.0 ppm and 7.5 ppm (7.28 ppm and 7.36-7.40 ppm). 2D TOCSYindicated a correlation between the 7.36-7.40 ppm peaks with rhamnoseCH₃ peaks (˜1.32 ppm; FIG. 7), suggesting those peaks (7.36-7.40 ppm)are proton signals of TSC derivatized 23B rhamnose. According to thenon-activated serotype 23B polysaccharide structure, the only possibleproton for 7.36-7.40 ppm peaks on the rhamnose ring is H3.

gCOSY data indicated the peaks at 5.46 ppm correlate with peaks at 7.28ppm. NOESY data indicated the peaks at 5.46 ppm are in close proximitywith the peak at 7.37 ppm (H3). Those data suggested the peaks at 5.46ppm belong to TSC derivatized rhamnose H1, and the peaks at 7.28 ppmbelong to TSC derivatized rhamnose H2, FIG. 8.

According to the above data, the main activation site for the serotype23B polysaccharide is at rhamnose C2/C3 position, as indicated in FIG.1B.

Example 3 Conjugation of S. Pneumoniae Serotype 23A Polysaccharide toCRM197 Using Reductive Amination in Dimethylsulfoxide

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in dimethyl sulfoxide (DMSO). Redissolved polysaccharide andCRM197 solutions were then combined and conjugated as described below.The resulting conjugate was purified by ultrafiltration prior to a final0.2-micron filtration. Several process parameters within each step, suchas pH, temperature, concentration, and time were controlled to yieldconjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was either size-reducedby acid hydrolysis or by homogenization. Acid hydrolysis was performedby adding acetic acid to 200 mM, incubating at 90° C. for 1.5 hours,then neutralizing by adding cold potassium phosphate pH 7 buffer to 400mM. For homogenization, pressure and number of passes through thehomogenizer were controlled to 800-1000 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 5 NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. The sodium metaperiodate addedwas 0.20-0.24 moles of sodium metaperiodate per mole of polysacchariderepeating unit to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit). Theoxidation reaction proceeded for 2 hours at 22° C.

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 5 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.0 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a concentration of 25-50 mM. The polysaccharide andCRM197 solutions were blended to achieve a polysaccharide concentrationof 1.8-3.0 g Ps/L (grams polysaccharide/liter) and a polysaccharide toCRM197 mass ratio of 1.5. The mass ratio was selected to control thepolysaccharide to CRM197 ratio in the resulting conjugate. Sodiumcyanoborohydride (1 mole per mole of polysaccharide repeating unit) wasadded and conjugation proceeded for 2-4 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 mole per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1-3 hoursat 22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. For somebatches, the batch was concentrated and diafiltered at approximately 4°C. against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, usinga 30 kD NMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diaftiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Table 1 shows the attributes of serotype 23A conjugate prepared in DMSO.

TABLE 1 Attributes of S. pneumoniae serotype 23A conjugate from DMSOconjugation Lysine Consumption Free Protein/ Oxidized Ps Conjugate(mol/mol Free Ps/ Total Mw Mw Ps:Pr CRM197) Total Ps Protein  97 kD 3837kD 1.20 11.8 0.2% 0.6% 190 kD 5620 kD 1.07 11.1 2.7% 3.0%

Example 4 Conjugation of S. Pneumoniae Serotype 23B Polysaccharides toCRM197 Using Reductive Amination in Dimethylsulfoxide

Polysaccharide was dissolved, sized to a target molecular mass,chemically activated and buffer-exchanged by ultrafiltration. Activatedpolysaccharide and purified CRM197 were individually lyophilized andredissolved in dimethyl sulfoxide (DMSO). Redissolved polysaccharide andCRM197 solutions were then combined and conjugated as described below.The resulting conjugate was purified by ultrafiltration prior to a final0.2-micron filtration. Several process parameters within each step, suchas pH, temperature, concentration, and time were controlled to yieldconjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation

Purified pneumococcal capsular Ps powder was dissolved in water and0.45-micron filtered. Dissolved polysaccharide was homogenized to reducethe molecular mass of the Ps. Homogenization pressure and number ofpasses through the homogenizer were controlled to 400 bar/5 passes.

Size-reduced polysaccharide was concentrated and diafiltered againstwater using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22° C. and pH 5 with asodium acetate buffer to minimize polysaccharide size reduction due toactivation. Polysaccharide activation was initiated with the addition ofa 100 mM sodium metaperiodate solution. Sodium metaperiodate added at0.10-0.13 moles of sodium metaperiodate per mole of polysacchariderepeating unit to achieve a target level of polysaccharide activation(moles aldehyde per mole of polysaccharide repeating unit).

The activated product was diafiltered against 10 mM potassium phosphate,pH 6.4 followed by diafiltration against water using a 10 kDa NMWCOtangential flow ultrafiltration membrane. Ultrafiltration was conductedat 2-8° C.

Polysaccharide Conjugation to CRM197

Purified CRM197, obtained through expression in Pseudomonas fluorescensas previously described (WO 2012/173876 A1), was diafiltered against 2mM phosphate, pH 7.0 buffer using a 5 kDa NMWCO tangential flowultrafiltration membrane and 0.2-micron filtered.

Activated polysaccharides were formulated for lyophilization at 6 mgPs/mL with sucrose concentration of 5% w/v. CRM197 was formulated forlyophilization at 6 mg Pr/mL with sucrose concentration of 1% w/v.

Formulated Ps and CRM197 solutions were individually lyophilized.Lyophilized Ps and CRM197 materials were redissolved individually inequal volumes of DMSO. The polysaccharide solution was spiked withsodium chloride to a final concentration of 0-50 mM. The polysaccharideand CRM197 solutions were blended to achieve a polysaccharideconcentration of 5.0 g Ps/L and a polysaccharide to CRM197 mass ratio of1.5. The mass ratio was selected to control the polysaccharide to CRM197ratio in the resulting conjugate. Sodium cyanoborohydride (1 mole permole of polysaccharide repeating unit) was added, and conjugationproceeded for 2-4 hours at 22° C.

Reduction with Sodium Borohydride

Sodium borohydride (2 mole per mole of polysaccharide repeating unit)was added following the conjugation reaction and incubated for 1 hour at22° C. The batch was diluted into 150 mM sodium chloride, withapproximately 0.025% (w/v) polysorbate 20, at approximately 4° C.Potassium phosphate buffer was then added to neutralize the pH. Thebatch was concentrated and diafiltered at approximately 4° C. against150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDNMWCO tangential flow ultrafiltration membrane.

Final Filtration and Product Storage

The batch was then concentrated and diafiltered against 10 mM histidinein 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered then diluted with additional10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015% (w/v)polysorbate 20, dispensed into aliquots and frozen at ≤−60° C.

Table 2 shows the attributes of S. pneumoniae serotype 23Bpolysaccharide conjugate prepared in DMSO.

TABLE 2 Attributes of S. pneumoniae serotype 23B polysaccharideconjugate from DMSO conjugation Lysine Consumption Free Protein/Oxidized Ps Conjugate (mol/mol Free Ps/ Total Mw Mw Ps:Pr CRM197) TotalPs Protein 179 kD 3299 kD 1.28 6.2 13% 5.1%

Example 5 Formulation of Monovalent Conjugates

Pneumococcal polysaccharide-CRM197 conjugates from serotypes 23A and 23Bwere prepared as described in Examples 3 and 4. Pneumococcalpolysaccharide-CRM197 conjugates from serotype 23F were prepared asdescribed in U.S. Pat. No. 8,192,746. The required volume of bulkconjugates needed to obtain the target concentration of individualserotypes were calculated based on batch volume and concentration ofindividual bulk polysaccharide concentrations. Bulk conjugates of the S.pneumoniae serotypes (23A, 23B, and 23F) were combined with excipients,sterile filtered and added to APA under mixing conditions. The finalconcentration of each monovalent conjugate vaccine was 4 μg/mL (w/vPnPs) with 20 mM Histidine, 150 mM NaCl, 0.2% (w/v) PS-20 and 0.250mg/mL (w/v Al) in the form of APA.

Example 6 Monovalent Conjugate New Zealand White Rabbit ImmunogenicityStudy

The immunogenicity of the monovalent conjugates was evaluated in a NewZealand White Rabbit (NZWR) model. Adult New Zealand White rabbits(NZWR, n=3/group) were intramuscularly (IM) immunized with 0.25 ml ofrespective monovalent conjugate vaccine on day 0 and day 14 (alternatingsides). Monovalent pneumococcal conjugate vaccine was dosed at 1 μg PnPs(S. pneumoniae serotype 23A or 23B polysaccharide each conjugated toCRM197) with 62.5 μg aluminum phosphate adjuvant (APA) per immunization.Sera were collected prior to study start (pre-immune) and on days 14(post-dose 1, PD1) and 28 (post-dose 2, PD2). NZWRs were observed atleast daily by trained animal care staff for any signs of illness ordistress. The vaccine formulations in NZWRs were deemed to be safe andwell tolerated. All animal experiments were performed in strictaccordance with the recommendations in the Guide for Care and Use ofLaboratory Animals of the National Institutes of Health. The NZWRexperimental protocol was approved by the Institutional Animal Care andUse Committees at both Merck & Co., Inc (Kenilworth, N.J.) and Covance(Denver, Pa.).

NZWR sera were tested in ELISA assays to evaluate IgG immunogenicityusing a 1-2 mg/ml respective PnPs coating concentration. Functionalantibody was determined through opsonophagocytosis assays (OPA) based onpreviously described protocols. See, e.g., Caro-Aguilar et al., 2017,Vaccine 35:865-72 and Burton et al., 2006, Clin Vaccine Immunol13(9):1004-9.

Monovalent pneumococcal polysaccharide conjugate vaccines from S.pneumoniae serotypes 23A and 23B were found to be immunogenic in rabbits(FIG. 9) and generate functional antibody which killed the respectivebacterial strain (FIG. 10).

Example 7 Monovalent Conjugate New Zealand White Rabbit ImmunogenicityStudy (23A, 23B, 23F Cross Protection)

Rabbits were immunized with 23A-CRM197/APA, 23B-CRM197/APA or23F-CRM197/APA to evaluate cross-reactivity between each S. pneumoniaeserotype.

Overall, rabbits immunized with S. pneumoniae serogroup 23 monovalentconjugate vaccines had the highest IgG and OPA titers to the homologouspolysaccharide and bacterial strain, respectively (FIGS. 11A-B, 12A-B,13A-B). 23A-CRM197/APA and 23F-CRM197/APA showed low/no cross-reactivityto S. pneumoniae 23B serogroup PnPs and bacterial strain compared torabbits immunized with the 23B-CRM197/APA conjugate (FIGS. 12A-B). UsingDunnett's multiple comparison tests, 23B-CRM197/APA immunized rabbitshad significantly higher IgG immunogenicity compared to 23A-CRM197/APAand 23F-CRM197/APA immunized rabbits (P=0.007 and 0.016, respectively)(FIG. 12A). Likewise, 23B-CRM197/APA immunized rabbits had significantlyhigher functional antibody compared to 23A-CRM197/APA and 23F-CRM197/APAimmunized rabbits (P=0.0002 and 0.002, respectively) (FIG. 12B). Tocover S. pneumoniae serogroup 23, a pneumococcal polysaccharideconjugate vaccine should comprise at least serotype 23A/23Fpolysaccharides and serotype 23B polysaccharide to protect against S.pneumoniae serotypes 23A, 23B, and 23F.

Example 8 Formulation of Pneumococcal Conjugate Vaccines for RabbitPolyvalent Study

A multivalent pneumococcal conjugate vaccine consisting of differentconjugate bulk blend preparations (including from S. pneumoniaeserotypes 16F, 23A, 23B, 24F and 31) was prepared using pneumococcalpolysaccharide-CRM197 conjugates and was formulated in 20 mM histidinepH 5.8 and 150 mM sodium chloride and 0.1% w/v polysorbate-20 (PS-20) at4 μg/mL each serotype for a total polysaccharide concentration of 84μg/mL. The conjugates were prepared by individually conjugating theCRM197 protein to pneumococcal polysaccharide (PnPs) types (includingfrom S. pneumoniae serotypes 16F, 23A, 23B, 24F and 31). The requiredvolume of bulk conjugates needed to obtain the target concentration ofindividual serotypes was calculated based on batch volume andconcentration of individual bulk polysaccharide concentrations. Theindividual conjugates were added to a solution of histidine, sodiumchloride and Polysorbate-20 (PS-20) to create the conjugate blend. Theformulation vessel containing the conjugate blend was mixed using amagnetic stir bar, and sterile filtered into another vessel. Theformulations were then filled into plastic syringes, glass syringes, orvials and stored at 2-8° C.

Example 9 Immunogenicity of a Multivalent Pneumococcal Conjugate Vaccinein New Zealand White Rabbits

Adult New Zealand White rabbits (NZWR, n=5/group) were intramuscularly(IM) immunized with 0.5 ml of the multivalent pneumococcal conjugatevaccine described in Example 8 on day 0 and day 14 (alternating sides).The multivalent pneumococcal conjugate vaccine was dosed at 2 μg of eachconjugated PnPs per immunization. Sera were collected prior to studystart (pre-immune) and on days 14 (post-dose 1, PD1) and 28 (post-dose2, PD2). NZWRs were observed at least daily by trained animal care stafffor any signs of illness or distress. The vaccine formulations in NZWRswere deemed to be safe and well tolerated. All animal experiments wereperformed in strict accordance with the recommendations in the Guide forCare and Use of Laboratory Animals of the National Institutes of Health.The NZWR experimental protocol was approved by the Institutional AnimalCare and Use Committees at both Merck & Co., Inc and Covance (Denver,Pa.).

NZWR sera were evaluated for IgG immunogenicity using a multiplexedelectrochemiluminescence (ECL) assay. This assay was developed for usewith rabbit serum based on the human assay described by Marchese et al.(Optimization and validation of a multiplex,electrochemiluminescence-based detection assay for the quantitation ofimmunoglobulin G serotype-specific antipneumococcal antibodies in humanserum. Clin Vaccine Immunol. 16(3): 387-96 (2009)) using technologydeveloped by MesoScale Discovery (a division of MesoScale Diagnostics,LLC, Gaithersburg, Md.) which utilizes a SULFO-TAG™ label that emitslight upon electrochemical stimulation. SULFO-TAG™-labeled anti-rabbitIgG was used as the secondary antibody for testing NZWR serum samples.Functional antibody was determined through multiplexed opsonophagocyticassays (MOPA) based on previously described protocols available onlineat the Bacterial Respiratory Pathogen Reference Laboratory at theUniversity of Alabama at Birmingham using Opsotiter® 3 software (UABResearch Foundation, Caro-Aguilar et al, 2017, supra, Burton et al.,2006, supra).

Polysaccharide-protein conjugates prepared from S. pneumoniae serotypes16F, 23A, 23B, 24F, and 31 in a multivalent pneumococcal conjugatevaccine were found to be immunogenic for both post dose 1 (PD1) and postdose 2 (PD2) in rabbits (FIG. 14). They also generated functionalantibody which killed vaccine-type bacterial strains (FIG. 15). Rabbitsimmunized with the multivalent pneumococcal conjugate vaccine at the 2μg dose had significantly higher PD1 MOPA titers for four serotypescompared to pre-immune rabbit sera (FIG. 15). Rabbits immunized with themultivalent pneumococcal conjugate vaccine at the 2 μg dose hadsignificantly higher PD2 MOPA titers for all five serotypes compared topre-immune rabbit sera (FIG. 15). Log Transformed data were analyzed byOne-way ANOVA with Dunnett's test to determine significance.

What is claimed is:
 1. A polysaccharide-carrier protein conjugate,wherein the polysaccharide has an S. pneumoniae serotype 23Bpolysaccharide repeating unit of the following structure:

wherein the carrier protein is CRM197, and further wherein the conjugatehas a molecular weight from 1,000 kDa to 10,000 kDa.
 2. Thepolysaccharide-carrier protein conjugate of claim 1, wherein thepolysaccharide-carrier protein conjugate has a polysaccharide to carrierprotein mass ratio from 0.4 to 2.0.
 3. The polysaccharide-carrierprotein conjugate of claim 1, wherein the carrier protein is conjugatedto the serotype 23B polysaccharide unit through the 2^(nd) or 3^(rd)carbon of the rhamnose sugar.
 4. The polysaccharide-carrier proteinconjugate of claim 1, wherein the polysaccharide has a molecular weightof between 50 kDa and 1,000 kDa.
 5. The polysaccharide-carrier proteinconjugate of claim 1, wherein the polysaccharide has a molecular weightof between 100 kDa and 800 kDa.
 6. The polysaccharide-carrier proteinconjugate of claim 1, wherein the polysaccharide has a molecular weightof between 100 kDa and 300 kDa.
 7. The polysaccharide-carrier proteinconjugate of claim 1, wherein the degree of conjugation of the conjugateis between 2 and
 15. 8. The polysaccharide-carrier protein conjugate ofclaim 1, wherein the degree of conjugation of the conjugate is between 8and
 12. 9. The polysaccharide-carrier protein conjugate of claim 1,wherein the polysaccharide-carrier protein conjugate has apolysaccharide to carrier protein mass ratio from 0.5 to 3.0.
 10. Thepolysaccharide-carrier protein conjugate of claim 1, wherein thepolysaccharide-carrier protein conjugate has a polysaccharide to carrierprotein mass ratio from 0.5 to 1.5.
 11. The polysaccharide-carrierprotein conjugate of claim 1, wherein the polysaccharide-carrier proteinconjugate has a polysaccharide to carrier protein mass ratio of about1.3.
 12. The polysaccharide-carrier protein conjugate of claim 1,wherein the polysaccharide-carrier protein conjugate comprises less thanabout 30% of non-covalently associated polysaccharide compared to thetotal amount of polysaccharide.
 13. The polysaccharide-carrier proteinconjugate of claim 1, wherein the polysaccharide-carrier proteinconjugate comprises less than about 25% of non-covalently associatedpolysaccharide compared to the total amount of polysaccharide.
 14. Thepolysaccharide-carrier protein conjugate of claim 1, wherein thepolysaccharide-carrier protein conjugate comprises less than about 20%of non-covalently associated polysaccharide compared to the total amountof polysaccharide.
 15. The polysaccharide-carrier protein conjugate ofclaim 1, wherein the polysaccharide-carrier protein conjugate comprisesless than about 15% of non-covalently associated polysaccharide comparedto the total amount of polysaccharide.
 16. The polysaccharide-carrierprotein conjugate of claim 1, wherein the polysaccharide has a molecularweight of between 100 kDa and 300 kDa; wherein the degree of conjugationof the conjugate is between 4 and 7; wherein the mass ratio ofpolysaccharide to carrier protein in the conjugate is between 0.5 and1.5; and wherein the conjugate comprises less than about 15% of freepolysaccharide compared to the total amount of polysaccharide.
 17. Apolysaccharide-carrier protein conjugate, wherein the polysaccharide hasan S. pneumoniae serotype 23B polysaccharide repeating unit of thefollowing structure:

wherein the carrier protein is CRM197; and further wherein the whereinthe polysaccharide-carrier protein conjugate has a polysaccharide tocarrier protein mass ratio from 0.5 to 3.0.
 18. Thepolysaccharide-carrier protein conjugate of claim 17, wherein thepolysaccharide-carrier protein conjugate has a polysaccharide to carrierprotein mass ratio from 0.5 to 1.5.
 19. The polysaccharide-carrierprotein conjugate of claim 17, wherein the polysaccharide-carrierprotein conjugate has a polysaccharide to carrier protein mass ratio ofabout 1.3.
 20. The polysaccharide-carrier protein conjugate of claim 17,wherein the carrier protein is conjugated to the serotype 23Bpolysaccharide unit through the 2^(nd) or 3^(rd) carbon of the rhamnosesugar.
 21. The polysaccharide-carrier protein conjugate of claim 17,wherein the polysaccharide has a molecular weight of between 50 kDa and1,000 kDa.
 22. The polysaccharide-carrier protein conjugate of claim 17,wherein the polysaccharide has a molecular weight of between 100 kDa and800 kDa.
 23. The polysaccharide-carrier protein conjugate of claim 17,wherein the polysaccharide has a molecular weight of between 100 kDa and300 kDa.
 24. The polysaccharide-carrier protein conjugate of claim 17,wherein the degree of conjugation of the conjugate is between 2 and 15.25. The polysaccharide-carrier protein conjugate of claim 17, whereinthe degree of conjugation of the conjugate is between 8 and
 12. 26. Thepolysaccharide-carrier protein conjugate of claim 17, wherein thepolysaccharide-carrier protein conjugate comprises less than about 30%of non-covalently associated polysaccharide compared to the total amountof polysaccharide.
 27. The polysaccharide-carrier protein conjugate ofclaim 17, wherein the polysaccharide-carrier protein conjugate comprisesless than about 25% of non-covalently associated polysaccharide comparedto the total amount of polysaccharide.
 28. The polysaccharide-carrierprotein conjugate of claim 17, wherein the polysaccharide-carrierprotein conjugate comprises less than about 20% of non-covalentlyassociated polysaccharide compared to the total amount ofpolysaccharide.
 29. The polysaccharide-carrier protein conjugate ofclaim 17, wherein the polysaccharide-carrier protein conjugate comprisesless than about 15% of non-covalently associated polysaccharide comparedto the total amount of polysaccharide.
 30. The polysaccharide-carrierprotein conjugate of claim 17, wherein the polysaccharide has amolecular weight of between 100 kDa and 300 kDa; wherein the degree ofconjugation of the conjugate is between 4 and 7; wherein the mass ratioof polysaccharide to carrier protein in the conjugate is between 0.5 and1.5; wherein the conjugate comprises less than about 15% of freepolysaccharide compared to the total amount of polysaccharide; andwherein the conjugate has a molecular weight from 1,000 kDa to 10,000kDa.